We have developed methodology whereby dendritic cells (DCs) are derived from precursors in small samples, 40-50 ml, of human blood. These DCs are unsually active in inducing T cell mediated immunity in tissue culture, but must be evaluated for efficacy in humans. In addition, the normal trafficking pattern of DCs in vivo is to move from tissues via the lymph to lymph nodes. Therefore different routes of DC administration (intradermal, subcutaneous, intramuscular) are important to test. By monitoring the lymph node draining the site of injection (palpation and ultrasound), one has a means of monitoring the immune response locally. We will evaluate DCs that have been expanded ex vivo from the precursors that are found in relatively small amounts of donor blood. The use of dendritic cells that are developing under defined conditions ex vivo also facilitates the loading of antigen presenting MHC (major histocompatibility complex) products with antigenic peptides, since the developing cells are synthesizing large amounts of these MHC products. Specifically, it is now possible to generate about 1.5-3 million, mature and active DCs from a 40-50 ml sample of normal blood [previously the best yield from an entire unit of blood was 106 DCs]. The method involves the culture of T cell-depleted blood cells with a mixture of recombinant human cytokines, especially GM-CSF and IL-4 for 6 days, after which the cells are matured for 5 more days in culture with an autologous monocyte conditioned medium. During the latter period the developing DCs are pulsed with an appropriate antigen. In the case of volunteers we will test the following 3 antigens. 1) Tetanus toxoid, the standard protein that is used to vaccinate against tetanus toxin, will be used to boost the primed T helper cells that are present in most volunteers. 2) Keyhole limpet hemocyanin, or KLH, to which most individuals are immunologically naive, will be used to test the capacity of the DCs to prime the immune system. 3) An influenza virus matrix peptide GILGFVFTL, will be used to boost the killer cell arm of the immune system, since this peptide is well known to be presented on MHC class I (HLA-2.1) molecules to CD8+ killer type T cells. Most of us are primed to influenza antigens, so that the recall CTL response to this virus provides an optimal control to determine if autologous DCs, pulsed ex vivo with viral antigens, can boost the CTL response in vivo. The peptide will be made according to Good Laboratory Practices [GLP] at Sloan Kettering Institute, where peptides have been synthesized previously for administration in humans.